Hybrid nanopores and uses thereof for detection of analytes

ABSTRACT

The invention relates to a hybrid structure including perforated solid substrate having at least one nanopore perforating therethrough, and devices and uses thereof.

TECHNOLOGICAL FIELD

The present disclosure relates to structures and devices comprisinghybrid nanopore structures and uses thereof.

BACKGROUND

Nanopore-based analysis has recently emerged as a promising toolenabling detection and analysis of analytes such as ions, nucleic acidmolecules, polypeptides and others during their translocation throughthe nanopore. Nanopores are generally classified into three main groups:(i) synthetic nanopores (ii) biological nanopores and (iii) combinationof biological and synthetic nanopores.

Hall A R et al [1] have inserted α-hemolysin into a solid-state nanoporeby attaching a long DNA molecule to the protein. In addition, it wassuggested to ratchet DNA through a nanopore in a controllable manner,e.g., phi29 molecular motor positioned on the entrance of the proteinnanopore [2-4] and electrostatic screening by manipulating the chargedistribution on the solid state nanopore wall [5-6].

WO11130312A1 [7] describes a nanopore device comprising a solid supportand a cyclic molecule attached effectively by a covalent linkage to azone on the interior sidewall surface of the channel.

Khoutorsky A et al [8] and WO2012/160565 [9 describe that SP1 and itsderivatives can be used to generate hydrophilic nanochannels in theplasma membrane of living cells.

Wang et al 2013 [10] describe a system composed of SP1 nanopores inlipid bilayers for determination of short single stranded nucleic acidsequences.

REFERENCES

-   [1] A. R. Hall, A. Scott, D. Rotem, K. K. Mehta, H. Bayley and C.    Dekker, Nat. Nanotechnol., 2010, 5, 874-877.-   [2] D. Wendell, P. Jing, J. Geng, V. Subramaniam, T J Lee, C.    Montemagno and P. X. Guo, Nat. Nanotechnol., 2009, 4, 765-772.-   [3] G. M. Cherf, K. R. Lieberman, H. Rashid, C. E. Lam, K. Karplus    and M. Akeson, Nature Biotechnology, 2012, 30, 344-348.-   [4] E. A. Manrao, I. M. Derrington, A. H. Laszlo, K. W.    Langford, M. K. Hopper, N. Gillgren, M. Pavlenok, M. Niederweis    and J. H. Gundlach, Nature Biotechnology, 2012, 30, 349-U174.-   [5] B. Q. Luan, H. B. Peng, S. Polonsky, S. Rossnagel, G.    Stolovitzky and G. Martyna, Phys. Rev. Lett., 2010, 104.-   [6] Y. H. He, M. Tsutsui, C. Fan, M. Taniguchi and T. Kawai, ACS    Nano, 2011, 5, 5509-5518.-   [7] WO11130312-   [8] A. Khoutorsky, A. Heyman, 0. Shoseyov, Me. Spica., Nano Lett.    2011, 11:2901-2904.-   [9] WO2012/160565-   [10] H. Y. Wang, Y. Li, L. X. Qin, A. Heyman, O. Shoseyov, I.    Willner, Y T. Long, H. Tian, Chem Commun 2013, 49(17): 1741-1743.-   [11] WO2002/070647-   [12] WO2004/022697-   [13] WO2007007325-   [14] WO2011/027342

SUMMARY OF THE INVENTION

The present invention is based on the development of a unique platformwhich enables diagnosis and measurement of one or more of presence,identity and quantity of chemical or biological entities (analytes) in asample. The unique platform of the invention is based on syntheticnanopores which are fabricated on a surface region of a device, which istuned for measuring a parameter associated with the analytes passingthrough the nanopores.

In accordance with the invention, the nanopores are decorated, orincorporated with, or comprised of, or coated by ring-like polypeptidessuch as SP1 polypeptide and any fragment, peptide, analogues, homologousand derivatives thereof. The central pore (hole) of the ring-likepolypeptide coincides and overlaps with the nanopore axis in such a waythat a continuous channel is formed, connecting an opening face of thering-like polypeptide and one opening of the nanopore. The continuouschannel comprising both the polypeptide and the nanopore is herebyreferred to as a hybrid nanopore.

The hybrid nanopores of the invention have been determined to exhibitdifferent characteristics than the bare nanopore, free of thepolypeptide. For example, as will be further demonstrated hereinbelow,it is shown that the translocation rate (time) of analytes through thehybrid nanopore was slower compared to the translocation rate throughbare synthetic nanopores. The presence of the ring-like structure, e.g.,SP1, in the hybrid nanopore not only affected the translocation rate(time), but also the conformation of the analyte during translocation.The hybrid nanopore was also found to selectively translocate a singleconformation of the analyte.

Thus, control and fine-tuning of parameters such as translocation rateand analyte conformation through the nanopore permit a sensitiveplatform that enables a superior temporal resolution during the passing(translocation) of materials through the nanopore.

Therefore, in accordance with a first aspect, the present inventionprovides a hybrid structure comprising (a) a solid substrate having atleast one nanopore perforating therethrough, and (b) at least onering-like polypeptide situated at a region of said at least onenanopore, said region being selected from an opening of the nanopore andan interior region of said nanopore.

The binding of the at least one ring-like polypeptide to the nanopore isdifferent than covalent binding. In some embodiments, the at least onenanopore comprises at its opening surface at least one ring-likepolypeptide. In other embodiments, the at least one ring-likepolypeptide is situated within the interior region of the nanopore.

According with the present disclosure, the solid substrate is the solidcontinuous material in which one or more nanopores are situated. Thethickness of the substrate defines the length or depth of the nanoporestructure. The solid substrate is characterized by having a first faceor surface and an opposite face or a second face or surface. Thedistance between the first and second faces defines the thickness of thesubstrate and the length or depth of the nanopore structure.

In other words, when referring to the first face and second face of thesubstrate, it should be referred to the planar surfaces of the substratethat are the faces (top end and/or bottom) of the substrate. In someembodiments, the first surface or face and the second surface or facemay be considered as parallel surfaces or substantially parallelsurfaces.

Once the nanopores are perforated through the substrate, from one faceto the other, the substrate may be referred to as a membrane.

When referring to the solid membrane, it should be noted that it doesnot encompass a cellular membrane or a bi-lipid layer membrane. In someembodiments, the solid substrate is synthetic. In some otherembodiments, the solid substrate is an inorganic sheet. In someembodiments, the solid substrate comprises a material selected fromsilicon, aluminum, titanium, hafnium, graphene, glass, quartz, diamondand teflon.

In some embodiments, the solid substrate is comprised or is of a dopedmaterial, such as doped silicon or doped diamond or any of the materialslisted above in doped forms.

In some other embodiments, the solid substrate is comprised or is of anundoped material, as listed above.

In some embodiments, the solid substrate is selected of a materialcomprising at least one of silicon nitride (SiN), silicon dioxide(SiO₂), aluminum oxide (Al₂O₃), titanium oxide (TiO₂) hafnium oxide(HfO₂) and graphene.

In some embodiments, the solid substrate consists or comprises siliconnitride.

The term “nanopore” as used herein denotes pores (holes, openings)present within the solid substrate, as defined. In the context of thepresent disclosure, the nanopores can be viewed as perforations havingeach a three dimensional representation of a channel (tunnel) with twoopenings at opposite sides of the solid substrate, wherein the twoopening are connected by an interior defined by a height (length,depth). It should be noted that the nanopore in the context of thepresent disclosure is present within the solid substrate such that oneof the nanopore openings is present at the first face of the substrateand the second opening is present at the second opposite face of thesubstrate.

The interior of the nanopore is spanned from one opening at the firstsurface (first opening) to an opening at the opposite surface (secondopening) and connecting the two openings. The interior may be an openinterior allowing flow throughout of any medium, e.g., a liquid mediumor any material.

The first opening and the second opening of the nanopore are eachcharacterized by a diameter that may be similar or different. Whenreferring to opposite faces of the nanopore, it is noted that the twoopenings may be considered as essentially parallel openings or nearlyparallel openings. In some embodiments, the two openings are co-axiallypositioned.

In some embodiments, each of the nanopores has, on average, a diameterof up to about 50 nm; in other embodiments, between about 1 nm to about50 nm; in further embodiments, between about 1 nm to about 20 nm; insome further embodiments, between about 2 nm to about 10 nm; in somefurther embodiments, between about 3 nm to about 8 nm and in some otherembodiments between 3 nm to 5 nm.

In some embodiments, the interior of the nanopore spanning the firstopening and the second opening has a length from about 5 nm to about 50nm; in some other embodiments, from about 10 nm to about 40 nm; in somefurther embodiments, from about 20 nm to about 35 nm.

The nanopores may be drilled in the solid substrate or alternatively maybe manufactured by any available method known in the art. Thefabrication of the nanopore(s) within the solid substrate may beachieved by any one or more of the following non-examples: feedbackcontrolled low energy (0.5-5.0 keV) noble gas ion beam sculpting,high-energy (200-300 keV) electron beam illumination. The nanoporeproperties, such as for example diameter and length, may be determinedby known methods in the field, such as transmission electron microscopy(TEM) and/or atomic force microscopy (AFM).

The solid substrate may comprise a plurality of nanopores. The pluralityof nanopores may be arranged in an array of nanopores, wherein in thearray the nanopores are arranged in groups or in a pattern, wherein eachgroup or pattern of nanopores being homogeneous or heterogeneous in atleast one parameter selected from nanopore density, nanopore size,nanopore depth and nanopore structure. For example, for certainapplications, one group of nanopores may have on average the same porediameter, while another group of nanopores is formed to have a differentpore diameter. In other cases, each group of nanopores may be formed tocomprise a plurality of nanopores having different pore diameter.

The hybrid structure described herein may be considered in accordancewith some embodiments as a self assembled structure. The termself-assembly or any lingual variation thereof denoted a process inwhich the components of the hybrid nanopore structure are assembled intoa structure (pattern) by a force which may or may not be imposed on thesystem to direct the assembly. In some embodiments, the process may beconsidered as being driven, without needing to introduce an externalforce. In some other embodiments, the process may use application ofvoltage. Thus, in line with some embodiments of the present invention,the hybrid structure may be considered as a self-assembled hybridstructure.

The ring-like polypeptide employed in accordance with the presentinvention is not limited to a specific protein and may be considered assuitable for any protein/polypeptide having a ring-like shape, namely anoligomeric polypeptide (protein) arranged in a circular ring shape(ring-like), having a central cavity (as inner hole) such that itscenter coincides with that of the nanopore. The ring-like polypeptide(protein) is further selected to form a stable interaction with thenanopore opening or the nanopore interior walls, and thus is selectednot to associate with the nanopore region (opening or interior walls)covalently, either directly or via a linker having functional groupsenabling the binding.

The ring-like polypeptide may be characterized by having an outerdiameter defining an outer rim of the ring-like structure polypeptideand an inner diameter defining the diameter of the inner cavity (asinner hole). The plane comprising the inner diameter is referred hereinas the X-Y plane and as such defines the planar orientation of thepolypeptide. The ring-like polypeptide may be further characterized byhaving a height referred herein as a Z axis. The ring like polypeptideaccording to the present disclosure is characterized by having asymmetric structure when viewed along the X-Y plane. In someembodiments, the ring-like polypeptide is not characterized by having amushroom like shape but rather a doughnut like shape.

In some embodiments, the at least one ring-like polypeptide has an innerdiameter of between about 1 nm to about 10 nm. In some otherembodiments, the at least one ring-like polypeptide has an innerdiameter of between about 1 nm to about 7 nm. In some furtherembodiments, the at least one ring-like polypeptide has an innerdiameter of between about 3 nm to about 4 nm.

In some embodiments, the at least one ring-like polypeptide has an outerdiameter of between about 8 nm to about 18 nm. In some otherembodiments, the at least one ring-like polypeptide has an outerdiameter of between about 10 nm to about 15 nm. In some furtherembodiments, the at least one ring-like polypeptide has an outerdiameter of between about 11 nm to about 13 nm.

The hybrid structure as described herein is characterized by having theinner diameter of the at least one ring-like polypeptide coincide withor match the nanopore diameter and as such, when viewed along the Zaxis, the centers of the inner diameter of the polypeptide and thediameter of the nanopore are essentially at the same location.

The location of the at least one ring like polypeptide within theinterior of the nanopore is dictated by a balance between the outerdiameter of the ring like polypeptide and the diameter of the nanopore.The ring-like polypeptide may have an outer diameter (namely thediameter of the entire polypeptide referred to herein as outer rim) thatis larger, equal or smaller than the diameter of the nanopore. In someembodiments, the diameter of at least one nanopore is smaller or equalto the outer diameter of the at least one ring-like polypeptide. In someembodiments, the outer diameter/rim of the polypeptide is larger thanthat of the nanopore and as such the polypeptide cannot enter theinterior of the nanopore while adopting a conformation at which thediameters centers are co-axially oriented.

In some embodiments, the at least one ring-like polypeptide is situatedon an opening of the nanopore. In connection with the presentdisclosure, the surface (face) associated with the polypeptide is afirst surface of the membrane. This can be viewed as at least onering-like polypeptide located on top of the first opening of thenanopore.

In some embodiments, the diameter of at least one nanopore is largerthan the outer diameter of the at least one ring-like polypeptide. Insome embodiments, the at least one ring-like polypeptide may be locatedwithin the nanopore interior surface.

The ring-like polypeptide may be selected to comprise several monomericsubunits, being either identical (homo) or different (hetero) from eachother, together forming a ring-like structure, e.g., complexpolypeptides. The polypeptide may be a native homo-oligomer orhetero-oligomer comprising monomeric subunits arranged, for example, ina concentric arrangement.

The ring-like polypeptide that may be used in accordance with theinvention may be a heat shock protein (HSP). The expression of HSP isincreased when cells are exposed to elevated temperatures and otherstress. The ring-like polypeptide may be for example HSP 60, HSP 70,HSP90 or thermolysin.

In some embodiments, the ring-like polypeptide is stable protein 1 (SP1)polypeptide [11-13].

The preparation, structural modification (mutations) and characteristicsof SP1 are disclosed in international patent application nos.WO2002/070647 [11], WO2004/022697 [12], WO2007/007325 [13] andWO2011/027342 [14], and corresponding US patent applications, each beingincorporated herein by reference.

As used herein “SP1” (stable protein 1) represents a homo-dodecameroligomeric protein having a ring-shape, referred also as a“doughnut-like” shape. SP1 polypeptide is naturally localized in thecytoplasm of plant cells and is not found in animal, e.g., mammalian(human or non-human) cells. SP1 has a net charge of −12 charge unitsthat are distributed on its surface.

When referring to SP1 it may be considered as consisting of 12 monomerswhich may be identical and as such having a molecular weight of 149 kDa.It is an extremely stable (T_(M)˜109° C.) and is considered as a boilingstable polypeptide, having a structural oligomeric stability followingtreatment at about 95° C., in an aqueous solution, for at least 10minutes, as determined by a size fractionation assay. In addition, SP1is characterized as a denaturant-stable polypeptide, having a structuraloligomeric stability of an oligomeric protein following treatment inaqueous solution containing 1:2,000 molar ratio (monomer:SDS), asdetermined by a size fractionation assay. SP1 polypeptide was shown tobe a functionally related protein that is involved in the folding andunfolding of other proteins.

In the context of the present disclosure, when referring to SP1 itshould be considered to encompass SP1 and any fragment, peptide,variant, analogues, homologue and derivatives thereof. Further, in thecontext of the present disclosure, when referring to SP1 and anyfragment, peptide, variant, analogues, homologue and derivatives thereofit should be considered to encompass also a hetero-dodecamer, namely apolypeptide having monomers with different sequences.

It should be noted that the different SEQ ID NOs provided herein defineamino acid sequences of one monomer of SP1 or nucleotide sequencesencoding one monomer of SP1.

In some embodiments, the SP1 is the wild type polypeptide (a monomer ofwhich disclosed, for example, in [14] as SEQ ID NO: 4; being referred toherein as SEQ ID NO: 1) or any fragment, peptide, variant, analogues,homologue and derivatives thereof disclosed, for example, in any ofreferences [11-14], each being incorporated herein by reference). Insome embodiments, the SP1 polypeptide is the wild type polypeptide (amonomer of which is disclosed, for example, in [14] as SEQ ID NO: 4being referred to herein as SEQ ID NO: 1) or any derivatives thereof(disclosed, for example, in any of references [11-14], each beingincorporated herein by reference).

It should be appreciated that in certain embodiments, SP1 protein refersto the SP1 polypeptide as denoted by SEQ ID NO: 1. As noted throughout,this refers to a sequence of one monomer of the SP1. Specifically, theSP1 protein comprises an amino acid sequence of one monomer of 108 aminoacid residues as denoted by GenBank Accession No. A.1276517.1.

In certain embodiments, the SP1 polypeptide comprises the amino acidsequence MATRTPKLVKHTLLTRFKDEITREQIDNYINDYTNLLDLIPSMKSFNWGTDLGAELNRGYTHAFESTFESKSGLQEYLDSAALAAFAEGFLPTLSQRLVIDYFLY as denoted by SEQ IDNO: 1.

In some embodiments, the SP1 polypeptide is a polypeptide encoded by thepolynucleotide deposited in NCBI under GenBank: AJ276517.1 (SEQ IDNO:8).

In some embodiments, the SP1 polypeptide is wild-type SP1 polypeptide(SEQ ID NO:1).

As may be appreciated by those versed in the art, genetic modificationof a polypeptide is of common practice and includes mutations in thepolynucleotide encoding the respective polypeptide, such that aselective mutation in the nucleotide sequence would result in a desiredamino acid mutation. Any mutation of the SP1 polypeptide referred toherein is a mutation based on the wild-type polypeptide. Mutations inthe polypeptide may include substitutions (mutations) of at least oneamino acid, deletion of at least one amino acid or addition of at leastone amino acid. Thus, the polypeptide used in accordance with thepresent invention may be selected from the wild-type SP1 polypeptide,cysteine mutated/substituted/added SP1 polypeptide, histidinemutated/substituted/added SP1 polypeptide and methioninemutated/substituted/added SP1 polypeptide.

Certain embodiments of the invention involve SP1 polypeptide and anyfragment, peptide, analogues, homologue and derivatives thereof. Itshould be appreciated that such peptides (polypeptides) or amino acidsequences are preferably isolated and purified molecules, as definedherein. The term “purified” or “isolated” refers to molecules, such asamino acid sequences, or peptides that are removed from their naturalenvironment, isolated or separated. An “isolated peptide” is therefore apurified amino acid sequence. “Substantially purified” molecules are atleast 60% free, preferably at least 75% free, and more preferably atleast 90% free from other components with which they are naturallyassociated. As used herein, the term “purified” or “to purify” alsorefers to the removal of contaminants from a sample.

The term “polypeptide” as used herein refers to amino acid residues,connected by peptide bonds. A polypeptide sequence is generally reportedfrom the N-terminal end containing free amino group to the C-terminalend containing free carboxyl group.

More specifically, “amino acid molecule”, “amino acid sequence” or“peptide sequence” is the order in which amino acid residues connectedby peptide bonds, lie in the chain in peptides and proteins. Thesequence is generally reported from the N-terminal end containing freeamino group to the C-terminal end containing amide. Amino acid sequenceis often called peptide, protein sequence if it represents the primarystructure of a protein, however one must discern between the terms“Amino acid sequence” or “peptide sequence” and “protein”, since aprotein is defined as an amino acid sequence folded into a specificthree-dimensional configuration and that had typically undergonepost-translational modifications, such as phosphorylation, acetylation,glycosylation, manosylation, amidation, carboxylation, sulfhydryl bondformation, cleavage and the like.

Amino acids, as used herein refer to naturally occurring and syntheticamino acids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. “Amino acidanalogs” refers to compounds that have the same fundamental chemicalstructure as a naturally occurring amino acid, i.e., an alpha carbonthat is bound to a hydrogen, a carboxyl group, an amino group, and an Rgroup, e.g., homoserine, norleucine, methionine sulfoxide, methioninemethyl sulfonium. Such analogs have modified R groups or modifiedpeptide backbones, but retain the same basic chemical structure as anaturally occurring amino acid. “Amino acid mimetics” refers to chemicalcompounds that have a structure that is different from the generalchemical structure of an amino acid, but that functions in a mannersimilar to a naturally occurring amino acid Amino acids may be referredto herein by either their commonly known three letter symbols or by theone-letter symbols recommended by the IUPAC-IUB Biochemical NomenclatureCommission.

It should be noted that in addition to any of the SP1 derived fragmentsor peptides encompassed herein, the invention further encompasses anyderivatives, analogues, variants or homologues of any of the SP1polypeptide. The term “derivative” is used to define amino acidsequences (polypeptide), with any insertions, deletions, substitutionsand modifications to the amino acid sequences (polypeptide). In someembodiments, these do not alter the activity of the originalpolypeptides. By the term “derivative” it is also referred tohomologues, variants and analogues thereof, as well as covalentmodifications of a polypeptides made according to the present invention.

In some embodiments, the SP1 polypeptide comprises additional histidineresidues (SEQ ID NO:2) and encoded by the polynucleotide having thesequence SEQ ID NO:9. More specifically, the SP1 polypeptide comprisethe amino acid sequenceMHHHHHHATRTPKLVKHTLLTRFKDEITREQIDNYINDYTNLLDLIPSMKSFNWGTDLGMESAELNRGYTHAFESTFESKSGLQEYLDSAALAAFAEGFLPTLSQRLV IDYFLY denoted asSEQ ID NO:2. As such, the 6His-SP1 derivative describes hereincorresponds to SEQ ID NO:2.

In further embodiments, the SP1 polypeptide is a homologous variant towild type SP1. The homologous variant may comprise for example deletionof amino acids in the N-terminal region of the wild type SP1.

In some embodiments, the SP1 polypeptide is a polypeptide with adeletion of amino acids in the N-terminal region. This variant SP1polypeptide having SEQ ID NO:3, is encoded by the polynucleotide havingthe sequence SEQ ID NO:10.

In some embodiments, the SP1 polypeptide comprises mutations of aminoacids to cysteine. Non-limiting examples of such polypeptides comprise:SEQ ID NO:4 (encoded by the polynucleotide having the sequence SEQ IDNO:11); SEQ ID NO:5 (encoded by the polynucleotide having the sequenceSEQ ID NO:12); SEQ ID NO:6 (encoded by the polynucleotide having thesequence SEQ ID NO:13); and SEQ ID NO:7 (encoded by the polynucleotidehaving the sequence SEQ ID NO:14).

In some other embodiments, the SP1 polypeptide comprises the amino acidsequence denoted as SEQ ID NO:4. In some further embodiments, the SP1polypeptide is a polypeptide encoded by the polynucleotide denoted bySEQ ID NO:11.

In some other embodiments, the SP1 polypeptide comprises the amino acidsequence denoted as SEQ ID NO:5. In some further embodiments, the SP1polypeptide is a polypeptide encoded by the polynucleotide denoted bySEQ ID NO:12.

In some other embodiments, the SP1 polypeptide comprises the amino acidsequence denoted as SEQ ID NO:6. In some further embodiments, the SP1polypeptide is a polypeptide encoded by the polynucleotide denoted bySEQ ID NO:13.

In some other embodiments, the SP1 polypeptide comprise the amino acidsequence denoted as SEQ ID NO:7. In some further embodiments, the SP1polypeptide is a polypeptide encoded by the polynucleotide denoted bySEQ ID NO:14.

In some other embodiments, the SP1 polypeptide comprises the amino acidsequence denoted as SEQ ID NO:15. In some further embodiments, the SP1polypeptide is a polypeptide encoded by the polynucleotide denoted bySEQ ID NO:16.

In some embodiments, the SP1 polypeptide comprises the amino acidsequence MRKLPDAATRTPKLVKHTLLTRFKDEITREQIDNYINDYTNLLDLIPSMKSFNWGTDLGMESAELNRGYTHAFESTFESKSGLQEYLDSAALAAFAEGFLPTLSQRLV IDYFLY denoted asSEQ ID NO:15.

In some other embodiments, the SP1 polypeptide comprises the amino acidsequence denoted as SEQ ID NO:17. In some further embodiments, the SP1polypeptide is a polypeptide encoded by the polynucleotide denoted bySEQ ID NO:18.

In some embodiments, the SP1 polypeptide comprises the amino acidsequence MRKLPDAATRTPKLVKHTLLTRFKDEITREQIDNYINDYTNLLDLIPSCKSFNWGTDLGMESAELNRGYTHAFESTFESKSGLQEYLDSAALAAFAEGFLPTLSQRLV IDYFLY denoted asSEQ ID NO:17.

In the context of the present disclosure, any reference to any one SEQID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ IDNO: 6, SEQ ID NO: 7, SEQ ID NO: 15 or SEQ ID NO: 17, is regarded as areference to one monomer of the dodecamer SP1 polypeptide. In addition,when referring to sequences denoted by SEQ ID NO: 8, SEQ ID NO: 9, SEQID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14,SEQ ID NO: 16 or SEQ ID NO: 18 it should be regarded as referring apolynucleotide encoding one monomer of the dodecamer SP1 polypeptide.

The SP1 may be genetically manipulated to comprise 12 monomers in whichat least one, at least two, at least three, at least four, at leastfive, at least six, at least seven, at least eight, at least nine, atleast ten, at least eleven or at least twelve monomers are the same ordifferent. In some embodiments, at least one, at least two, at leastthree, at least four, at least five, at least six, at least seven, atleast eight, at least nine, at least ten, at least eleven or at leasttwelve of the monomers are independently selected from SEQ ID NO: 1, SEQID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ IDNO: 7, SEQ ID NO: 15 and SEQ ID NO: 17.

The polypeptides employed in accordance with the invention may beproduced synthetically, or by recombinant DNA technology. Methods forproducing polypeptides peptides are well known in the art.

In some embodiments, the SP1 derivatives include, but are not limitedto, polypeptides that differ in one or more amino acids in their overallsequence from the polypeptides defined herein (either the SP1 protein orany fragment or peptide derived therefrom according to the invention),polypeptides that have deletions, substitutions, inversions oradditions.

In some embodiments, the derivatives are polypeptides, which differ fromthe polypeptides specifically defined in the present invention byinsertions of amino acid residues. It should be appreciated that by theterms “insertions” or “deletions it is meant any addition or deletion,respectively, of amino acid residues to the polypeptides, of between 1to 50 amino acid residues, between 20 to 1 amino acid residues, andspecifically, between 1 to 10 amino acid residues. Insertions ordeletions may be of any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 aminoacids and may occur at any position of the modified peptide, as well asin any of the N′ or C′ termini thereof.

The peptides of the invention may all be positively charged, negativelycharged or neutral. In addition, they may be in the form of a dimer, amultimer or in a constrained conformation, which can be attained byinternal bridges, short-range cyclizations, extension or other chemicalmodifications.

The polypeptides of the invention can be coupled (conjugated) throughany of their residues to another peptide or agent. For example, thepolypeptides of the invention can be coupled through their N-terminus toa lauryl-cysteine (LC) residue and/or through their C-terminus to acysteine (C) residue.

Further, the peptides may be extended at the N-terminus and/orC-terminus thereof with various identical or different amino acidresidues. As an example for such extension, the peptide may be extendedat the N-terminus and/or C-terminus thereof with identical or differentamino acid residue/s, which may be naturally occurring or syntheticamino acid residue/s. An additional example for such an extension may beprovided by peptides extended both at the N-terminus and/or C-terminusthereof with a cysteine residue. Naturally, such an extension may leadto a constrained conformation due to Cys-Cys cyclization resulting fromthe formation of a disulfide bond. Another example may be theincorporation of an N-terminal lysyl-palmitoyl tail, the lysine servingas linker and the palmitic acid as a hydrophobic anchor. In addition,the peptides may be extended by aromatic amino acid residue/s, which maybe naturally occurring or synthetic amino acid residue/s, for example, aspecific aromatic amino acid residue may be tryptophan. The peptides maybe extended at the N-terminus and/or C-terminus thereof with variousidentical or different organic moieties, which are not naturallyoccurring or synthetic amino acids. As an example for such extension,the peptide may be extended at the N-terminus and/or C-terminus thereofwith an N-acetyl group.

For every single peptide sequence defined herein, this inventionincludes the corresponding retro-inverse sequence wherein the directionof the peptide chain has been inverted and wherein all the amino acidsbelong to the D-series.

The invention also encompasses any homologues of the polypeptides(either the SP1 protein or any fragments or peptides thereof)specifically defined by their amino acid sequence according to theinvention. The term “homologues” is used to define amino acid sequences(polypeptide) which maintain a minimal homology to the amino acidsequences defined by the invention, e.g. preferably have at least about65%, more preferably at least about 75%, even more preferably at leastabout 85%, most preferably at least about 95% overall sequence homologywith the amino acid sequence of any of the polypeptide as structurallydefined above, e.g. of a specified sequence, more specifically, an aminoacid sequence of the polypeptides as denoted by any one of SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6,SEQ ID NO: 7, SEQ ID NO: 15, SEQ ID NO: 17.

More specifically, “homology” with respect to a native polypeptide andits functional derivative is defined herein as the percentage of aminoacid residues in the candidate sequence that are identical with theresidues of a corresponding native polypeptide, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent homology, and not considering any conservative substitutions aspart of the sequence identity. Neither N-nor C-terminal extensions norinsertions or deletions shall be construed as reducing identity orhomology. Methods and computer programs for the alignment are well knownin the art.

In some embodiments, the present invention also encompasses polypeptideswhich are variants of, or analogues to, the polypeptides specificallydefined in the invention by their amino acid sequence. With respect toamino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to peptide, polypeptide, orprotein sequence thereby altering, adding or deleting a single aminoacid or a small percentage of amino acids in the encoded sequence is a“conservatively modified variant”, where the alteration results in thesubstitution of an amino acid with a chemically similar amino acid.

Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologues, and alleles and analogous peptides of the invention.

For example, substitutions may be made wherein an aliphatic amino acid(G, A, I, L, or V) is substituted with another member of the group, orsubstitution such as the substitution of one polar residue for another,such as arginine for lysine, glutamic for aspartic acid, or glutaminefor asparagine. Each of the following eight groups contains otherexemplary amino acids that are conservative substitutions for oneanother:

-   -   1) Alanine (A), Glycine (G);    -   2) Aspartic acid (D), Glutamic acid (E);    -   3) Asparagine (N), Glutamine (Q);    -   4) Arginine (R), Lysine (K);    -   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);    -   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);    -   7) Serine (S), Threonine (T); and    -   8) Cysteine (C), Methionine (M)

More specifically, amino acid “substitutions” may be the result ofreplacing one amino acid with another amino acid having similarstructural and/or chemical properties, i.e., conservative amino acidreplacements Amino acid substitutions may be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphiphathic nature of the residues involved.For example, nonpolar “hydrophobic” amino acids are selected from thegroup consisting of Valine (V), Isoleucine (I), Leucine (L), Methionine(M), Phenylalanine (F), Tryptophan (W), Cysteine (C), Alanine (A),Tyrosine (Y), Histidine (H), Threonine (T), Serine (S), Proline (P),Glycine (G), Arginine (R) and Lysine (K); “polar” amino acids areselected from the group consisting of Arginine (R), Lysine (K), Asparticacid (D), Glutamic acid (E), Asparagine (N), Glutamine (Q); “positivelycharged” amino acids are selected form the group consisting of Arginine(R), Lysine (K) and Histidine (H) and wherein “acidic” amino acids areselected from the group consisting of Aspartic acid (D), Asparagine (N),Glutamic acid (E) and Glutamine (Q).

The derivatives of any of the polypeptides according to the presentinvention, e.g. of a specified sequence of any one of the polypeptidesdenoted by SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:15 or SEQ ID NO:17 may vary intheir size and may comprise the full length polypeptide or any fragmentthereof. In certain embodiments, the polypeptides may comprise one ormore amino acid residue surrogate. An “amino acid residue surrogate” asherein defined is an amino acid residue or peptide employed to producemimetics of critical function domains of peptides.

Examples of amino acid surrogate include, but are not limited tochemical modifications and derivatives of amino acids, stereoisomers andmodifications of naturally occurring amino acids, non-protein aminoacids, post-translationally modified amino acids, enzymatically modifiedamino acids, and the like. Examples also include dimers or multimers ofpeptides. An amino acid surrogate may also include any modification madein a side chain moiety of an amino acid. This thus includes the sidechain moiety present in naturally occurring amino acids, side chainmoieties in modified naturally occurring amino acids, such asglycosylated amino acids. It further includes side chain moieties instereoisomers and modifications of naturally occurring protein aminoacids, non-protein amino acids, post-translationally modified aminoacids, enzymatically synthesized amino acids, derivatized amino acids,constructs or structures designed to mimic amino acids, and the like.

It should be appreciated that the invention further encompass any of thepeptides, any serogates thereof, any salt, base, ester or amide thereof,any enantiomer, stereoisomer or disterioisomer thereof, or anycombination or mixture thereof. Pharmaceutically acceptable saltsinclude salts of acidic or basic groups present in compounds of theinvention. Pharmaceutically acceptable acid addition salts include, butare not limited to, hydrochloride, hydrobromide, hydroiodide, nitrate,sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate,lactate, salicylate, citrate, tartrate, pantothenate, bitartrate,ascorbate, succinate, maleate, gentisinate, fumarate, gluconate,glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate,ethanesulfonate, benzensulfonate, p-toluenesulfonate and pamoate (i.e.,1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts. Certain compounds ofthe invention can form pharmaceutically acceptable salts with variousamino acids. Suitable base salts include, but are not limited to,aluminum, calcium, lithium, magnesium, potassium, sodium, zinc, anddiethanolamine salts.

It should be noted that the invention further encompasses anypeptidomimetic compound mimicking the polypeptide of the invention,namely SP1 and any fragment or peptide thereof. When referring topeptidomimetics, what is meant is a compound that mimics theconformation and desirable features of a particular natural peptide butavoids the undesirable features, e.g., flexibility and bond breakdown.From chemical point of view, peptidomimetics can have a structurewithout any peptide bonds; nevertheless, the compound is peptidomimeticdue to its chemical properties and not due to chemical structure.Peptidoinimetics (both peptide and non-peptidyl analogues) may haveimproved properties (e.g., decreased proteolysis, increased retention orincreased bioavailability). It should be noted that peptidomimetics mayor may not have similar two-dimensional chemical structures, but sharecommon three-dimensional structural features and geometry. Eachpeptidomimetic may further have one or more unique additional bindingelements.

The hybrid nanopore structure, as defined herein, may be prepared by aprocess comprising contacting a solid substrate having at least onenanopore perforated therethrough with at least one ring-like polypeptideunder conditions permitting placement of said at least one ring-likepolypeptide at the rim opening of said at least one nanopore or in aninterior region of said at least one nanopore.

In some embodiments, the hybrid structure is formed by self-assembly. Insome embodiments, the formation of the hybrid structure is driven byapplication of voltage.

As shown in Example 2 herein below, and specifically in FIG. 2A,addition of SP1 to a solid substrate resulted in trapping of SP1 ontothe nanopore rim as indicted by the distinct three peaks. Further, asshown in FIGS. 2B to 2D, arrangement of SP1 was found to be in anorientation that was essentially parallel to the nanopore planarorientation. The trapping (coating) of SP1 in the nanopore was found tobe reversible and depended on the applied polarity.

As further shown in Example 2, specifically in FIGS. 2G and 2H, thetrapping of the SP1 in the nanopore may be affected by the SP1characteristics. For example, different derivatives of SP1 were found tohave different trapping behavior. Specifically, in an SP1 mutant, SiSP1,a lower voltage was required in order to trap the polypeptide in thenanopore.

The hybrid structure may be part of a device, e.g., an electronicdevice. The electronic device may comprise a measuring unit.

Thus, in some embodiments, the present invention provides a devicecomprising (i) a hybrid structure as defined herein and (ii) a measuringunit.

According to some embodiments, the hybrid structure is placed within thedevice such that it may separate two chambers. The two chambers may beheld in place separately. In some embodiments, and as described hereinbelow, the two chambers may be connected electrically only by theelectrolyte solution. The chambers as described herein may be preparedby any conventional material known in the art to be suitable for such apurpose. Non-limiting examples include polydimethylsiloxane (PDMS),plastic, teflon and any known insolating solid material. In someembodiments, the chambers comprise an electrolyte solution.

The device described herein may comprise an electrode assemblyconstructed of a set of at least two electrodes. In some embodiments,each chamber is equipped with an electrode or an electrode assembly. Insome embodiments, the electrode is an Ag/AgCl electrode.

As noted above, the at least one ring-like polypeptide is not limited toany polypeptide and may be for example SP1 as defined herein. In someembodiments, the device is configured such that the bottom of one of thetwo separated chambers faces (in connection with, touching upon) thefirst surface (with the SP1) and comprising the opening of the nanoporein the first surface. In some embodiments, this is referred as the cischamber (or cis reservoir).

In some embodiments, the chamber facing the first surface has an openingof between about 1 nm to about 10 nm; in some other embodiments, betweenabout 2 nm to about 5 nm; in some further embodiments, between about 3nm to about 4 nm. The opening is on top of the hybrid structure suchthat it covers the nanopore diameter. The opening in the chamber may beof any shape for example a funnel shape aperture. The opening in thechamber may be aligned with the hybrid nanopore by any known method inthe art, for example by an optical microscope.

In some other embodiments, the top of one of the two separated chambersfaces (in connection with, touching upon) the second opposite surfaceand comprising the opening of the nanopore in the second surface. Insome embodiments, this is referred as the trans chamber (or transreservoir).

In some embodiments and specifically when the chambers are filled withan electrolyte solution, flow of solution may be permitted through thenanopore from the first opening to the second opening via the interiorof the nanopore. Thus, the two separate chambers are in liquid or gascommunication.

In some embodiments, the device comprises a microfluidic system enablingchanging sample solution. In some embodiments, the device comprises acooling-heating system to control the temperature of the device. Thesesystems and any additional system used in the device may be manually orcontrolled by a computer. In some yet other embodiments, and in order toreduce possible noise, the device may be placed within a Faraday cageand even on top of a vibration isolation table.

The device according with the present disclosure comprises a measuringunit. The measuring unit is adapted to measure ionic current through thenanopore. In some embodiments, the ionic current is generated andmeasured by the same unit. In some other embodiments, different unitsare required to generate and measure the current. In some embodiments,the unit may be a voltage source, patch clamp system. In someembodiments, the generating and/or measuring unit may be furtherequipped with an amplifier and/or a low pass filter and/or digitizer.

In some embodiments, the measuring unit comprises a computer readablesystem.

The hybrid structure and the device comprising the structure may be usedfor analyzing a sample when the sample is placed in close proximity tothe nanopore or alternatively in the cis chamber and provided that thesample is allowed to pass through the nanopore. The sensitivity andspecificity of the hybrid nanopore described herein to monitortranslocation of analytes was determined herein.

Generally and as shown herein, when voltage is applied to the hybridnanopore and no analyte is presented near the nanopore or in the cischamber, a stable ionic current representing an open pore current may bemeasured.

When an analyte is added near the nanopore or to the cis chamber nearthe nanopore, the analyte may pass through the nanopore to the otherside of the nanopore (membrane), at time may be the trans chamber. Inaddition, the analyte may be present near one opening of the nanopore.When the analyte is present near (namely not in the interior) or insidethe nanopore (in the interior) (referred herein below also as themonitoring step), part of the ionic flow in the nanopore is changedcausing a detectable change in ionic current. The change may be anincrease in current or blockade in current. This signal (transient) maybe dependent on different parameters for example the properties of thenanopore, electrolyte solution, and the passing molecule. Thus, thehybrid nanopore provides a fundamental tool for sample analysis.

Thus, the present disclosure provides in accordance with its furtheraspect a method for analysis of at least one analyte in a samplecomprising: (a) applying a sample comprising at least one analyte orsuspected to comprise at least one analyte onto a hybrid structure,wherein the hybrid structure as defined herein. In accordance with thepresent disclosure the polypeptide is SP1 polypeptide. The next step (b)comprises permitting the sample to flow through the nanopore. In thesubsequent step (c), a determination is made of at least one of (i)presence or absence of an analyte in the sample, (ii) identity of theanalyte in the sample, and (iii) concentration of the analyte in thesample.

In accordance with the method described herein, the sample comprising atleast one analyte or suspected to comprise at least one analyte may bemixed with SP1 and then applied onto the solid nanopore.

The method may further comprise monitoring at least one measurableparameter related to the nanopore that may be indicative inter alia ofthe passing of an analyte through the pore and thus permit thedetermination step (c).

In some embodiments, at least one measurable parameter is a chemical ora physical parameter. In some further embodiments and as detailed hereinbelow, the at least one measurable parameter is an optical parameter. Insome embodiments, the measurable parameter is an electrical signal.

Several measurable parameters may be obtained when an analyte is near orin a hybrid nanopore. In some embodiments, a change in the current (orthe current value) may be detected.

As used herein, the “change in the current value” may be determined(measured) by comparing an observed current to a current measured at anearlier time point, e.g., in the absence of a sample, and determiningthe ratio of the values between the two measurements. The change in thecurrent may be either a blockage or an increase in the current. In someembodiments, a blockage (drop) in the current may be observed and, e.g.,subsequently compared to a previous measurement.

In some embodiments, the change in current may be expressed as thefraction or percentage of the open nanopore current, open channelcurrent, I/Io, where I is the blockade current and Io is the openchannel current (e.g., in case an analyte is not detected). In someembodiments, the current blockade as noted above may indicate that ananalyte is present at a region proximal to hybrid nanopore or in thenanopore structure, e.g., during passage through the hybrid nanoporechannel.

In some embodiments, the change in current may be defined as an eventhaving measurable time duration. The time duration of the change in thecurrent or the time duration of a measurable or observed or detectedevent refers to the period over which the change in current occurs(measurable in millisecond, seconds, etc). In some embodiments, themeasured time of the change (event) may reflect on the translocationtime (passing) of a sample or an analyte, as defined herein, through thehybrid structure.

In some embodiments, the period over which the change in the currentoccurs may be determined as the time difference between a time pointwhen a first current change (increase or blockage) is observed and alater time point when the change is arrested or further altered. In someembodiments, the time period is measured until a further change in theblockage or increase in the current is observed. This may be usuallydetermined over a threshold value that is set beyond the baseline noiselevel.

In some embodiments, the time duration of the change may be fitted byGaussian. In some other embodiments, the time duration of the change maybe fitted by exponential with time constant.

In some other embodiments, the events are represented by transientspikes (indicative of one or more change in a measurable current). Insome other embodiments, the frequency of current change events may bedetermined.

In some embodiments, the event integral, as described herein may bedetermined by calculating the integral of ionic current over theduration of an event.

In some embodiments, the at least one measurable parameter is at leastone of (i) change in current, and (ii) time duration of a change in thecurrent and any combination thereof.

When referring to electric current it should be noted to encompasselectric current either in a direction parallel to the surface of thesolid membrane; or tunneling current perpendicular to the surface.

In some embodiments, the at least one parameter may be determinedmanually by visual inspection or by automated means or any combinationof the two. In some embodiments, automated means including computationalanalysis may be used, for example by application of appropriatealgorithms.

In some embodiments, the at least one measured parameter may be used toobtain at least one data value related to the at least one parameter.

In some embodiments the method comprise comparing the at least onemeasurable parameter and/or the at least one data value with apredetermined standard corresponding parameter and/or corresponding datavalue. This may be done by referring to known measurements or toexperiments wherein no measurement is done prior to application of thesample to be tested.

In some other embodiments, the method comprises comparing the at leastone measurable parameter and/or the at least one data value with acorresponding parameter and/or corresponding data value obtained in acontrol study the absence of a sample before application of a sample.

As used herein the term “comparing” denotes any examination of the atleast one measurable parameter and/or the at least one data valueobtained in the samples as detailed throughout in order to discoversimilarities or differences between at least two different samples. Itshould be noted that comparing according to the present inventionencompasses the possibility to use a computer based approach.

The present method disclosed herein also comprises steps performed priorto application of the sample to be tested onto the hybrid structure. Insome embodiments, the method further comprises filling the two separatechambers, the cis chamber and trans chamber with an electrolytesolution. In some embodiments, the solution is a standard solution, forexample comprising 1 M KCl TE (10 mM tris and 1 mM EDTA at pH 7.4)buffer solution. In some other embodiments, after filling the chamberswith the electrolyte solution, initial testing of the nanopore isperformed, for example such as nanopore conductivity testing. In somefurther embodiments, these initial measurements may be used to determineat least one measurable parameter related to the nanopore possibly todetermine data value obtained in the absence of a sample. In somefurther embodiments, voltage is applied to the electrodes and thecurrent of the nanopore is determined in the absence of a sample, toobtain the open channel current (also referred herein as the opennanopore current).

In connection with the present disclosure, the sample is applied ontothe hybrid nanopore. Application of the sample may be by any methodknown in the filed for example pouring, injecting and others, enablingthe sample to be in close proximity and in physical contact to oneopening of the nanopore.

In some embodiments, the sample is applied onto the hybrid nanopore, assuch the sample is applied to the nanopore's opening comprising the SP1polypeptide. In some specific embodiments, the sample is applied ontothe first opening of the hybrid nanopore within the first surface of themembrane.

In some embodiments, the sample comprising the SP1 is applied onto thenanopore (with no SP1 or with partial SP1 coating). In some specificembodiments, the sample is applied onto the first opening of thenanopore within the first surface of the membrane.

The sample is then permitted to pass through the nanopore. In thecontext of the present disclosure, passing of a sample through thenanopore enables measuring at least one measurable parameter related tothe nanopore. In some embodiments, the at least one measurableparameters may be at least one physical or chemical property. In someother embodiments, the at least one measurable parameters may be atleast one optical property.

Without being bound by theory, it is suggested that in case an analyteis present in the sample, it will induce a change in at least one of themeasured parameters (measurable parameter). Further, and without beingbound by theory, in case an analyte is not present in the sample, nochange in the at least one parameter is induced. More specifically, nochange in the current is detected.

Generally, when used, the term “change” or “difference” of at least oneparameter relates to an increase or decrease in at least one parameteras described herein. More specifically, a reduction in the current is byat least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%,14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%,28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%,42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%,56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or about1000% as compared to corresponding current in a control measurement (attimes open nanopore current).

In some embodiments, the method provides a qualitative (yes=presence,no=absence) result by comparison with a control measurement as describedherein. It is to be understood that the term “absence” in the context ofthe assay also encompasses presence of the analyte in an amount that islower than the detection limit of the method.

The “sample” according to the present invention may be any sampleincluding, but not limited to, biological samples obtained frombiological systems (including cell cultures, micro-organism cultures),biological samples obtained from subjects (including humans andanimals), samples obtained from the environment for example soilsamples, water samples, agriculture samples (including plant and cropsamples), food samples. The term “sample” may also include body fluidssuch as whole blood sample, blood cells, bone marrow, lymph fluid,serum, plasma, urine, sputum, saliva, faeces, semen, spinal fluid orCSF, the external secretions of the skin, respiratory, intestinal, andgenitourinary tracts, tears, milk, any human organ or tissue, anybiopsy, for example, lymph node or spleen biopsies, any sample takenfrom any tissue or tissue extract, any sample obtained by lavageoptionally of the breast ductal system, plural effusion, samples of invitro or ex vivo cell culture and cell culture constituents.

In some embodiments, the sample is a liquid sample. In some embodiments,the liquid sample is liquid in its natural state. In some furtherembodiments, the liquid sample is pre-treated to be in a liquid state.Pre-treatment may be by any method that changes a sample that is notliquid in its natural state into a liquid state. In some embodiments,pre-treatment is by extraction. In some other embodiments, the samplecomprises at least one liquid fraction.

Depending on the specific method and results to be obtained, the samplein the context of the invention may be prepared prior to the analysis inin vitro settings and not necessarily obtained from a subject.

The term “analyte” as used herein denotes a molecule or an ion which maybe found in a sample, and which detection or quantification is required.In some embodiments, the sample may comprise a binding agent capable ofbinding to the analyte prior to or during passing through the nanopore(or hybrid nanopore). The term “binding agent” as used herein refers toany molecule capable of specifically binding to the analyte for examplean aptamer, an antibody, a receptor ligand or a molecular imprintedpolymer.

In some embodiments, the analyte may be a protein, a polypeptide, apeptide, a ganglioside, a lipid, a phospholipid, a carbohydrate, a smallmolecule or a nucleic acid.

Non-limiting examples in accordance with the invention are solublecancer markers, inflammation-associated markers, hormones, cytokines,drugs, and soluble molecules derived from a virus, a bacteria or afungus for example, toxins or allergens.

In some embodiments, the analyte is a cancer (or tumor) marker or aviral marker (or any fragment thereof). In general, a tumor marker maybe found in the body fluids such as in blood or urine, or in bodytissues. Tumor markers may be expressed or over expressed in cancer andare generally indicative of a particular disease process.

In some embodiments, the analyte is a nucleic acid.

In some embodiments, the analyte may be modified. In some embodiments,the analyte may be conjugated (chemically) to a moiety that may be anycompound capable of producing a detectable signal. The moiety may be forexample a chromophore, a fluorophore or a luminancephore. In some otherembodiments, the at least one measurable parameter may be an opticalsignal. As appreciated, Alkaline Phosphatase (AP) or Horse RadishPeroxidase (HRP) substrate detection may be achieved by chromaticsignal, fluorescence signal or luminescence signal, which may bedetected using various spectrophotometers and fluorometers.

In accordance with the present invention and as disclosed herein below,the analyte may be a nucleic acid molecule and in some embodiments ofthe present disclosure a modified nucleic acid molecule.

As shown in Example 3 herein, ds-DNA translocated through the nanopore.Interestingly, the ds-DNA passing through the hybrid nanopore adopts alinear confirmation as compared to the bare nanopore (no SP1). Furtherinterestingly, translocation through the nanopore was found to slower ascompared to the bare nanopore as indicated by the longer dwell time.

Without being bound by theory, it is suggested that the positive chargesthat are present in the inner pore of the SP1, may electrostaticallyinteract with the negatively charged DNA when translocated through itand as such slow down the DNA translocation. Previous studies showedthat DNA translocation through this hybrid structure was found to be thesame as when the α-hemolysin is buried in the less stable lipid bilayer.Namely, no advantage was observed to the hybrid nanopore. As such, theresults shown here are superior to the data found in the art.

An improvement of the translocation temporal resolution to fewnucleotides per ms surprisingly shown herein is highly advantage andpaves the way to a new arena of using hybrid nanopore with SP1 fornucleic acid sequencing. Further, the selectivity of the hybrid nanoporedisclosed herein for linear conformation is almost essential forsequencing the translocated nucleotides.

Thus, in accordance with another aspect, the present disclosure providesa method for sequencing a nucleic acid molecule comprising (a) applyinga sample comprising at least one nucleic acid molecule onto a hybridstructure, and determining the sequence of the nucleic acid molecule.

As used herein, “nucleic acid(s)” is interchangeable with the term“polynucleotide(s)” and generally refers to any polyribonucleotide orpoly-deoxyribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA or any combination thereof. “Nucleic acids” include, withoutlimitation, single- and double-stranded nucleic acids.

As used herein, the term “nucleic acid(s)” also includes DNAs or RNAs asdescribed herein that may contain one or more modified bases. Thus, DNAsor RNAs with backbones modified for stability or for other reasons are“nucleic acids”. The term “nucleic acids” as it is used herein embracessuch chemically, enzymatically or metabolically modified forms ofnucleic acids, as well as the chemical forms of DNA and RNAcharacteristic of viruses and cells, including for example, simple andcomplex cells. A “nucleic acid” or “nucleic acid sequence” may alsoinclude regions of single- or double-stranded RNA or DNA or anycombinations.

In some embodiments, the nucleic acid is DNA. In some other embodiments,the nucleic acid is RNA. In some embodiments, the nucleic acid is adouble stranded (ds) nucleic acid. In some other embodiments, thenucleic acid is a single stranded (ss) nucleic acid.

When referring to sequencing of at least one nucleic acid molecule, itshould be noted that the molecule may be a ds-DNA, ss-DNA, ds-RNA orss-RNA. The nucleic acid may be a synthetic molecule or alternatively anucleic acid molecule obtained from any biological sample, food sampleor the like as described herein. In some embodiments, the nucleic acidsubjected to analysis is in a linear conformation. In some furtherembodiments, the nucleic acid is an unstructured nucleic acid.

As noted herein, the nucleic acid may be a modified nucleic acid. Insome embodiments, the nucleic acid molecule contains 2-aminoadenosine,2-thiothymidine, inosine, and pyrrolopyrimidine. In accordance with someother embodiments, the nucleic acid molecule may be attached to afluorescent moiety. In accordance with some further embodiments thenucleic acid is biotinylated. As appreciated, modification of a nucleicacid as described herein may be by covalent bonding. The terms“conjugation”, “association”, “connection”, “interactions” are usedinterchangeably to denote a bonding between two chemical entities. Thebonding may be for example covalent binding, hydrogen binding,electrostatic binding, hydrophobic interactions and the like.

In some embodiments, the conformation of a nucleic acid molecule can beevaluated by the blockage of the current. As shown herein, translocationof a nucleic acid in a linear conformation has a distinct pattern thatdiffers from translocation of a variety of conformations.

In some other embodiments, the number of bases (also termed hereinnucleotides) or the size of a DNA molecule can be estimated from theintegrated area of an event. In some further embodiments, a linearnucleic acid molecule can be used as a marker.

In the context of the present disclosure evaluation of the nucleic acidconformation and the nucleic acid size may be determined for example byrecording events from a known nucleic acid (ladder) molecule, washingthe known molecule and adding a tested molecule.

As appreciated, detection of modified nucleic acid may be for example byoptical means such as fluorescence.

In some embodiments, in case the nucleic acid is ss-DNA or ss-RNA, thecomplementary strand may be formed prior to or during passing throughthe nanopore. In such embodiments, the method further comprisesproviding a suitable enzyme such as polymerase or exonuclease.

Sequencing of a nucleic acid molecule involves determining the preciseorder of nucleotides (also denoted herein as bases) within a nucleicacid molecule, namely, determine the order of the four bases: adenine,guanine, cytosine, and thymine in a strand of DNA or adenine, guanine,cytosine, and uracil in a strand of RNA.

In the context of the present disclosure, the sequencing methodcomprises passing of the nucleic acid molecule through the nanopore fromone opening to the opposite opening in a sequentialnucleotide-by-nucleotide translocation. The information of eachnucleotide is monitored separately in a monitoring step. In someembodiments, the monitoring step comprises determining at least onemeasurable parameter related to a nucleotide to possibly obtain a datavalue. The at least one measurable parameter and/or the at least onedata value may be indicative of a nucleotide. The at least onemeasurable parameter and/or data value is further compared to acorresponding predetermined standard parameter and/or corresponding datavalue or to a corresponding predetermined standard parameter and/orcorresponding data value obtained from a control nucleotide.

In some embodiments, the at least one measurable parameter and/or datavalue obtained at each monitoring step is compared to a predeterminedstandard parameter or data value of a nucleotides in a control sample.The resemblance of the at least one parameter or data value obtained ateach monitoring step to at least one parameter or data value obtainedfor any one of the different nucleotides in a control sample iscalculated in order to determine the nucleotide within the nucleic acidmolecule. The degree of resemblance is indicative of an existence of aspecific nucleotide in the nucleic acid molecule.

As such, the method according to some embodiments provides a qualitativetest by providing the analysis of the identity of the analyte and insome embodiments, the identity of the specific nucleotide within thenucleic acid molecule.

In another aspect, the present invention provides a method for thediagnosis of a condition in a subject comprising using an analysismethod in accordance with the invention as described above. In someembodiments, the analyte is an analyte associated with the condition andwherein the presence or absence of analyte is indicative of the presenceof a condition in the subject.

In another aspect, the present invention provides a method formonitoring the efficiency of a therapeutic regimen in a subjectsuffering from a condition comprising using an analysis method inaccordance with the invention as described above. In some embodiments,the analyte is associated with the condition and wherein the amount ofanalyte is indicative of the level of the condition and thereby of theefficiency of the therapeutic regimen in the subject.

In some embodiments, a sample is obtained from a subject and issubsequently subjected to a method according to the invention.

As used herein, “disease”, “disorder”, “condition” and the like, as theyrelate to a subject's health, are used interchangeably and have meaningsascribed to each and all such terms. In some embodiments, the conditionis a pathological condition.

The “pathological condition” according to the present invention may beselected from but not limited to cancer, inflammation, blood coagulationdisorders, and autoimmunity. Accordingly, the method of the inventionmay be used in the detection of known cancer markers, markers ofinflammation, such as procalcitonin which is a known marker for sepsis,peptides such as penicillin-binding protein 2 (PBP2), kinesin spindleprotein (KSP), toxins and allergens.

In some other embodiments, the pathological condition is a viral,bacterial or fungal infection. Non-limiting examples of viral infectioncomprises Hepatitis B virus (HBV), hepatitis C virus (HCV),Cytomegalovirus (CMV), Human immunodeficiency virus (HIV), Epstein-Barrvirus (EBV), HERPES virus, Polio virus, and influenza virus.

Non-limiting examples of bacterial infection comprises Listeria,Diphtheria, E. coli, Group B streptococcus (GBS), Group A streptococcus,Tuberculosis (TB), Salmonella, Vibrio Cholerae, Campylobacter,Brucellosis, meningococcus, Streptococcus pneumonia and Candida.

In some embodiments, the pathological condition is cancer. Cancer isinterchangeably used with the terms malignancy, tumor and is referred toherein as a class of diseases in which a group of cells displayuncontrolled growth and invasion that may destroy adjacent tissues, andsometimes leads to metastasis (spreading to other locations in thebody). Cancer may be a solid cancer or a non-solid cancer and may beclassified as carcinoma, sarcoma, lymphoma, leukemia, germ cell tumor,or blastoma.

In some further embodiments, the pathological condition is an autoimmunedisease. As appreciated in the art, autoimmune diseases arise from anoveractive immune response of the body against substances and tissuesnormally present in the body. Non limiting examples of autoimmunedisease are Multiple sclerosis, Arthritis, Autoimmune hepatitis, Crohn'sdisease, Diabetes mellitus type 1, Inflammatory bowel disease, Multiplesclerosis, Psoriasis, Rheumatoid arthritis, Wegener's granulomatosis.

Using the detection methods of the present invention the level of targetmolecules indicative of the pathological state may be determined.Therefore, the measurement of the levels of these target molecules canserve to diagnose the pathological condition, to monitor diseaseprogression and to monitor efficacy of a therapeutic regiment, i.e.monitor the response of the subject to treatment.

In some further embodiments, the condition is a non-pathologicalcondition.

Other aspects of the present invention provide use of the hybridstructure as disclosed herein in the manufacture of a device accordingto the invention.

In some other aspects, there is provided a hybrid structure and/ordevice comprising same for use in research purposes. Non-limitingexamples include laboratory use, scientific experiments and the like.

In some further aspect, there is provided a hybrid structure and/ordevice comprising same for use in analysis of at least one analyte in asample. In some embodiments, the hybrid structure is used in determiningat least one of (i) presence or absence of an analyte in the sample,(ii) identity of the analyte in the sample, (iii) concentration of theanalyte in the sample.

In accordance with the present disclosure, the hybrid structure is usedin sequencing a nucleic acid molecule

The term “about” as used herein indicates values that may deviate up to1 percent, more specifically 5 percent, more specifically 10 percent,more specifically 15 percent, and in some cases up to 20 percent higheror lower than the value referred to, the deviation range includinginteger values, and, if applicable, non-integer values as well,constituting a continuous range.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to better understand the subject matter that is disclosedherein and to exemplify how it may be carried out in practice,embodiments will now be described, by way of non-limiting example only,with reference to the accompanying drawings, in which:

FIGS. 1A-1D show SP1 translocation through solid nanopores.

FIG. 1A is a schematic illustration of translocation of SP1 through a 15nm solid-state nanopore in a SiN membrane (shown in the TEM image, thescale bar is 5 nm) separated between two reservoirs with a potentialdifference.

FIG. 1B shows four consecutive current versus time traces of SP1(concentration is 10 mg/mL) translocation through the solid-statenanopore at various voltages, translocation events were represented bytransient spikes, inset is a control experiment after diluting theprotein concentration, showing lower translocation events rate.

FIG. 1C is a graph showing the events rate dependence on the appliedvoltage with a logarithmic behavior. The dashed line showed a linearfitting (in log scale) to the experimental data (dots with error barsfrom at least 3 repetitions) at various voltages.

FIG. 1D shows SP1 translocation trace demonstrating the negative chargenature of the SP1, where translocation events were only observed atpositive polarity.

FIGS. 2A-2I show SP1 trapping on a solid nanopore.

FIG. 2A is a cartoon illustrating trapping of SP1 on top of a solidnanopore of 3 nm (smaller than the protein/polypeptide size), scale barin the TEM image is 5 nm, the typical trace of successful SP1 trappingon the pore above shows three distinct peaks in the current histogram,which represent open pore without protein (1), landing of protein on thepore (2), and final trapping (3), respectively at 400 mV.

FIGS. 2B-2D are traces of SP1 trapping in nanopores with variousdiameters (3 nm 5 nm and 8 nm, respectively) at 400 mV, with thebaseline current rises as the nanopore diameter increases. The scale barfor the TEM images is 5 nm.

FIG. 2E is a graph showing the dependence of the current reduction(ratio between current drop and baseline current) of the trapping events(current drop longer than 1 s is defined as an SP1 trapping event) onthe pore diameter measured for nanopores with 3, 5 and 8 nm diameter.Inset: shows the conductance model of nanopore taking the geometricalshape into account. The theoretical blocking values are based on thecircumstances of SP1 sitting on top of the pore (dots).

FIG. 2F shows traces of SP1 protein trapped on the nanopore and releasedby manually changing the polarity. When the polarity is switched back,another protein can be trapped.

FIG. 2G shows two traces at 200 and 400 mV of the trapping efficiency ofthe SP1 (L81C) protein on the nanopore (3 nm pore). A 400 mV potentialdifference is needed to trap the SP1 (L81C) protein.

FIG. 2H shows three traces at 200, 300 and 400 mV indicating anincreased trapping efficiency of a silicon-binding SP1 (SiSP1) mutant onthe nanopore (5 nm pore). For this mutant a potential difference of 200mV was sufficient to trap the silicon-binding SP1 protein on thenanopore.

FIG. 2I shows a trace demonstrating that SiSP1 can sometimes still betrapped after changing to negative voltage bias.

FIGS. 3A-3G show DNA translocation in SP1 hybrid nanopore.

FIG. 3A is a cartoon illustrating trapping of SP1 followed by DNAtranslocation, which reveals the fourth stage caused by DNAtranslocation through the hybrid pore. A typical trace demonstrates DNAtranslocation through L81CSP1 nanopore, recorded at 400 mV. The currenthistogram shows a fourth peak that represents DNA translocation.

FIG. 3B is a graph showing the 48 kbp dsDNA translocations rate throughSP1 hybrid nanopore as a function of DNA concentration at 400 mV.

FIGS. 3C and 3D shows insertion of 10 nm gold nanoparticle (GNP)connected by thiol to a 26 bases long ssDNA which is hybridized toanother 100 bases long ssDNA, into a hybrid nanopore. FIG. 3C: trappingof SP1 occurs after 1 second, resulting in the first reduction of thecurrent. After adding GNP-DNA conjugates, an insertion of GNP-DNA to theSP1 happens after 158 seconds, resulting in the second reduction of thecurrent to less than 10% of the baseline current. FIG. 3D: GNP-DNAconjugate stays in the SP1 pore for ˜1 s. Then, the GNP was released orhybridized 100 bases long ssDNA was dehybridized and GNP released,leading to a recovery of the open SP1 current.

FIGS. 3E and 3F show representative traces of DNA translocation in bareSiN (FIG. 3E) and SP1 nanopores (FIG. 3F). The events detection is doneusing adaptive threshold method with exclusion of short pulses (<0.1ms).

FIG. 3G shows a scatter plot and histogram of the dwell time andconductance of 48 kbp DNA translocated through L81SP1 hybrid nanopore(dots) and through bare SiN nanopore (triangles). The conductancehistogram shows a narrow peak for DNA translocation through L81SP1nanopore indicating a single DNA conformation within the L81SP1 withrespect to multiple peaks for DNA translocation through the bare SiNnanopore, indicating variable DNA conformations.

FIGS. 4A-4B are graphs showing that DNA translocation is slower throughthe SP1 hybrid nanopore.

FIG. 4A 48 kbp dsDNA translocation dwell time of SP1 hybrid nanoporesvs. bare solid state pore. The 400 mV trace was measured for SP1 (L81C)hybrid nanopores and the 100, 200, and 300 mV traces were measured forSiSP1 hybrid nanopores.

FIG. 4B depicts most probable dwell time as a function of voltage. Bothpanels show the clear slow down of the translocation by the SP1 protein.

DETAILED DESCRIPTION OF EMBODIMENTS Non-Limiting Examples MethodsNanopore Fabrication

Nanopores were fabricated in 30 nm thick, low-stress SiN windows (50×50μm²) supported by a silicon chip (Protochips) using a focused electronbeam of a 200 keV TEM (Tecnai, F20 G²). Nanopores with small sizes, suchas 3-4 nm were made using a shrinkage process by defocusing the electronbeam. Once the pores were drilled, they were stored in ethanol:ddH₂O(1:1, v:v) immediately to avoid any contamination.

Protein Synthesis

Protein expression, purification and refolding were carried out asdescribed in detail by Heyman et al. Briefly, E. Coli strain BL21(DE3)was used for protein expression, using IPTG (isopropylβ-d-thiogalactopyranoside) as inducer. The protein, which accumulated ininclusion bodies (IBs), was separated by centrifugation, washed,denatured and finally refolded to allow the self-assembly into itsdodecamer form. Further purification was conducted using ion-exchangechromatography method. Two types of SP1 mutants were used in this work:L81CSP1 (with no specific binding to Si) and SiSP1 (with specificbinding to Si).

The monomer of L81CSP1 is denoted herein as SEQ ID NO:5 and the monomersequences of the SiSP1 are denoted herein as SEQ ID NO:15 or SEQ IDNO:17.

Translocation Experiments

Nanopore membranes were treated in a Plasma Cleaner for 30 s tofacilitate wetting before being mounted in a custom electrophoresis flowcell (Nanopore Solution, Inc.). Two reservoirs on each side with avolume of 1 mL (trans and cis) were filled with filtered and degassedbuffer of 1M KCl, 10 mM Tris pH 7.4, 10% Glycerol, and 1 mM EDTA.

A pair of Ag/AgCl pellet electrodes was immersed in the two reservoirsand connected to an Axopatch 200B amplifier (Molecular Devices, Inc.) torecord ionic current flow through the nanopore. The whole setup was putin a double Faraday cage to lower external electrostatic interference.Signals were collected at 100 kHz sampling rate using a Digidata 1440A(Molecular Devices, Inc.) and filtered at 10 kHz using the built-in lowpass Bessel filter of Axopatch. Clampfit 10.2 (Molecular Devices, Inc.)was used for event detection with an adaptive threshold method, by whichshort pulses (<0.01 ms) are excluded. All the DNA samples were purchasedfrom Fermentas, Inc. and used as received. The DNA and the SP1 wereinjected into the cis-side, which is connected to the ground electrode,with a pipette and mixed well by repeatedly sucking and injecting withthe pipette.

Results

Example 1 Translocation of SP1 Through Nanopores

This example describes characterization of the translocation nature ofSP1 through nanopores. The SP1 has a net charge of −6 charge units permonomer that are distributed on its surface.

FIG. 1A illustrates a pore, slightly larger than the SP1 proteindiameter, in a SiN membrane, that separates two ionic buffer reservoirsequipped with a pair of electrodes with a potential difference.

The protein translocation was detected by measuring the transientcurrent blockage (on the time scale of microsecond to millisecond) whenthe protein was passing through a single nanopore under electrophoreticforce. FIG. 1B shows four consecutive 1 min traces at four differentvoltages (20, 50, 80, and 100 mV). An increased frequency of events wasobserved upon voltage raising, corresponding to an increasedtranslocation events rate, as expected when increasing the ionic currentand force acting on the charged proteins.

The observed rates at 20, 50, 80, and 100 mV were 51, 290±33, 465±32,587±125 min⁻¹, respectively. According to previous studies, the capturerate of polymers threading through a confined hole should follow anexponential increase with increasing voltage, represented by the Van'tHoff-Arrhenius relationship, R_(capture) αe^(V).

This experimental data obtained from 50 mV to 150 mV fitted thisrelationship (FIG. 1C). At 20 mV the translocation rate did not followthe above equation and was down shifted. It was thus suggested that at20 mV, the translocation might be dominated by diffusion. As can be seenin FIG. 1B (inset), dilution of the SP1 concentration led to deceasedrate.

In addition, SP1 translocation events were not observed when thepolarity was changed to negative bias but returned after it was changedback to positive bias (FIG. 1D). This suggested that SP1 is negativelycharged under these buffer conditions (1M KCl, pH 7.4) and was moved tothe nanopore and translocated through it upon positive bias application.This observation was consistent with the fact that the isoelectric point(pI) of the SP1 protein is 4.7.

The voltage and concentration dependence of the translocation confirmSP1 translocation through the nanopore and consequently also the abilityto bring the SP1 to the nanopore by dielectrophoresis.

Example 2 Trapping of SP1 in Nanopores

To trap SP1 on the nanopore, 3 nm to 8 nm diameter nanopores (mostly 3nm to 5 nm) (16 pores) were used (FIG. 2A). The trace in FIG. 2Ademonstrated a typical trapping process, which includes threeconsecutive steps, manifested by three distinct peaks in the currenthistogram provided on the right side of the trace.

These peaks were interpreted as follows: Initially, there were manyevents that reflected SP1 hitting on the nanopore, appearing as spikeson the baseline (peak 1). These were followed by an intermediateblockage, possibly in some tilted protein orientations (peak 2), andeventually by a successful trapping (peak 3) in an orientation parallelto the membrane surface.

DNA translocation through the SP1, shown later, was unlikely innon-planar SP1 orientation as the 3 nm width of the “doughnut-shape” SP1would totally block the 3 nm nanopore opening and not enable dsDNAtranslocation.

A simple equivalent electric circuit assumed that the SP1 poreintroduced a constant serial resistance to the ionic current in additionto the SiN pore resistance. Based on this model, the relative currentreduction after trapping should show a strong dependence on the porediameter. The experimental data fitted well with the calculated currentdrop assuming that SP1 was indeed sitting on top of the nanopore (FIGS.2B-2D).

FIGS. 2B-2D indicted that the relative current reduction in a hybridnanopore, i.e. after SP1 trapping in the nanopore was dependent on thenanopore diameter. The relative current reductions were observed to be45±7% (5 pores, 30 trapping events), 30±5% (2 pores, 25 trappingevents), and 10±2% (2 pores, 15 trapping events) for nanopores withdiameters of 3 nm, 5 nm and 8 nm, respectively, as shown in FIG. 2E.

According to the conductance model of nanopore the conductance of thenanopore could be described as

${G = {\sigma \left\lbrack {\frac{4\; h}{\pi \; d^{2}} + \frac{1}{D}} \right\rbrack}^{- 1}},$

where h is the thickness of the SiN membrane (30 nm), d is the averagediameter of the pore, a is the bulk conductivity of 1 M KCl solution(11.2 S m⁻¹), and D is the diameter of the pore opening. Theexperimental data fitted with the calculated current drop (56%, 27% and25% for nanopores with diameters of 3, 5 and 8 nm, respectively).

This suggested that SP1 is sitting on top of the nanopore where D and hwill be affected by SP1. Thus, the experimental data fitted well withthe calculated current drop suggesting that SP1 is sitting atop on thenanopore (FIGS. 2B-2E)

As shown in FIG. 2F, after the SP1 was trapped (left part of the trace),the polarity was changes to intentionally release the SP1 (marked by anarrow) and the baseline went back to the bare pore current level (namelywithout any protein trapped in the pore).

Thereafter another trapping event took place (right part of the trace).It should be noted that SP1 can be trapped as is, due to its naturallynegatively charged surface, namely without any further geneticmodifications. This is unlike α-hemolysin that needs was chemicallymodified for this purpose.

To demonstrate the variability of the trapping behavior and bindingaffinity to the surface, two SP1 mutants were compared, the first beingL81CSP1 (with no specific binding to Si) and the second being SiSP1 thathas Si-binding peptide in each N-termini that facilitates binding to Sisurfaces.

The results are shown in FIGS. 2G and 2H. As can be seen in FIG. 2G, forL81CSP1, the threshold voltage to trap SP1 on the nanopore was 400 mV.At lower potential of 200 mV, no trapping events were detected.

When SiSP1 was used, the trapping threshold was reduced to 200 mV (FIG.2H). As the voltage rises, the SiSP1 trapping frequency was furtherincreased.

As shown in FIG. 2I, changing polarity failed to release the tightlybound SiSP1 from the nanopore.

Comparison of the two protein mutants showed the influence of thegenetically engineered mutations on the protein trapping behavior andfurther confirms the trapping of the SP1 on top of the nanopore. Thiswas not observed for SP1 with no specific binding, in such case the SP1was released upon voltage polarity change.

Example 3 Translocation of DNA Through SP1-Dressed Nanopores

After characterizing the SP1 trapping, DNA translocation through anSP1-dressed nanopore was demonstrated (FIGS. 3A-3G).

The first three stages in the scheme shown in FIG. 3A were similar tothe SP1 trapping described above. After mixing SP1 proteins and dsDNAmolecules in the cis chamber, another set of blocking events wasobserved along the trapped SP1 level.

These additional events were attributed to DNA translocation through thehybrid SP1-SiN nanopore. In addition, their events frequency wasincreased as the DNA concentration was increased (FIG. 3B).

To further verify that the DNA was translocated through the nanopore, 10nm gold nanoparticle conjugated to 26 bp single-stranded DNA hybridizedto 100 bp single-stranded DNA was added to the solution and translocatedinto the hybrid nanopore, resulting in a much deeper (drop to 10-30% ofthe baseline) blockage of the SP1 pore by the nanoparticle (FIG. 3C).

Such clogging could last for seconds unless the conjugated DNA isdehybridized or dissociated from the gold nanoparticle by the electricalforce (FIG. 3D).

This further strengthens the suggestions that SP1 was sitting atop theSiN nanopore in a horizontal manner. These experiments confirm thatdsDNA is indeed passing through the SP1-dressed nanopore and that the4th peak in the histogram in FIG. 3A corresponds to dsDNA translocation.

FIGS. 3E and 3F show a comparison between translocation of X-DNA, 48kbp, through a bare nanopore (FIG. 3E) and through the hybrid, SP1dressed, nanopore (FIG. 3F). The uniform blocking level of the events inthe hybrid, SP1 dressed, nanopore trace demonstrated that dsDNA which istranslocated through the hybrid nanopore has a single conformation,likely linear, as opposed to DNA translocation through the bare solidstate nanopore that shows multiple conformations.

FIG. 3G provides a summary n comparison, using a scatter plot andhistograms, of the conductance and dwell time of 48 kbp X-DNAtranslocated through L81SP1 hybrid nanopore (dots) and through bare SiNnanopore (triangle).

The conductance histogram showed a single narrow peak for DNAtranslocation through the hybrid nanopore, suggesting a single linearDNA conformation with respect to multiple peaks for DNA translocationthrough the bare nanopore, suggesting variable DNA conformations.

dsDNA translocation through the hybrid nanopore blocks the ion currentwith a lower amplitude compared to dsDNA that is translocated through abare nanopore (FIGS. 3E, 3F and 3G).

When the DNA is translocated through the bare nanopore in its linearform, it caused a reduction of ˜2 ns in the ion conductance.

For the hybrid nanopore, the reduction in the blocking conductance to˜0.5 ns (FIGS. 3E, 3F and 3G) may be related to charge screening by theintrinsic positive charge in the inner pore of the SP 1.

Without being bound by theory, such a charge screening can lead to areduced ion flow, resulting in lower blocking conductance.

In addition, the DNA translocation dwell time through the hybridnanopore is longer by over an order of magnitude than the dwell time oftranslocation through the bare nanopore, as observed by monitoring thekinetics of the DNA translocation through the hybrid nanopore (FIG. 3Gand FIG. 4).

Without being bound by theory, it was suggested that the slowing down inthe dwell time through the hybrid nanopore may result from either theintrinsic positive charge residing in the inner pore of the SP1 or froma possible higher friction. These two parameters that can be controlledby using genetically or chemically modified SP1.

Since electrophoretic dragging of the DNA through the pore was thekinetic driving force, it was expected to obtain an exponentialdependence of dwell time on the voltage.

FIG. 4A demonstrates the dwell time distribution of DNA translocationmeasured at various driving voltages from 100, 200 and 300 mV for theSiSP1 and at 400 mV for L81CSP1.

The peak of the distribution is the most probable dwell time fortranslocation and is plotted in FIG. 4B.

For both bare and hybrid nanopores, an exponential dependence of thedwell time on the voltage was observed, which is in agreement withelectrophoretic-force driven translocation.

These results suggested that dsDNA is translocated through the trappedSP1. Compared to bare nanopores, the DNA translocation was slowed downby at least 10 fold for all the voltages (FIG. 4). This suggested thatthe absolute translocation velocity of dsDNA was dominated by theinteraction between the protein and the DNA translocating through thepore.

The hybrid nanopore was shown to have at least three central advantagesover the bare solid state nanopore.

First it enabled to slow down the translocation by over an order ofmagnitude. A further slow down might be achieved by genetic or chemicalmodification of the SP1 protein, thus addressing one of the centralchallenges on the track to DNA sequencing. An analysis that does nottake into account very fast hitting attempts of the DNA on the SP1 astranslocation events provides a further relative slowdown, up to nearlytwo orders of magnitude. The validity of such analysis must be, however,further controlled and verified.

Secondly, it allows translocation in a linear conformation only, unlikethe bare solid state nanopore, where numerous conformations are observedthat distort the translocation pattern shape and affect thetranslocation dwell time and naturally also the ability to sequence thetranslocated nucleotides.

The third advantage of the SP1 protein is its readiness for geneticengineering and functional modifications, in addition to its extremestability.

1-79. (canceled)
 80. A hybrid structure comprising (a) a solid substratehaving at least one nanopore perforating therethrough, and (b) at leastone ring-like polypeptide situated at a region of said at least onenanopore, said region being selected from an opening of the nanopore andan interior region of said nanopore, wherein the binding of the at leastone ring-like polypeptide to the nanopore being different from covalentbinding.
 81. The hybrid structure according to claim 80, wherein the atleast one nanopore having an opening at a first surface of the substrateand a further opening at an opposite surface of the substrate, andinterior surface spanning the opening at a first surface and the furtheropening at the opposite surface.
 82. The hybrid structure according toclaim 80, wherein the at least one ring-like polypeptide being situatedat an opening of the nanopore at a first surface of the substrate. 83.The hybrid structure according to claim 80, wherein at least onering-like polypeptide is selected from the group consisting of HeatShock Protein (HSP) 60, HSP70, HSP90, HSP100, thermolysine, stableprotein 1 (SP1) and any fragment, peptide, variant, analogues, homologueand derivatives thereof.
 84. The hybrid structure according to claim 83,wherein the ring-like polypeptide is stable protein 1 (SP1).
 85. Thehybrid structure according to claim 80, wherein the SP1 polypeptide is ahomo-dodecamer or hetero-dodecamer oligomeric polypeptide.
 86. Thehybrid structure according to claim 80, wherein at least one of the SP1polypeptide monomers is wild type SP1 polypeptide having an amino acidsequence denoted by SEQ ID NO:1.
 87. The hybrid structure according toclaim 86, wherein at least one of the SP1 polypeptide monomers isencoded by the polynucleotide deposited in NCBI under GenBank:AJ276517.1 (SEQ ID NO:8).
 88. The hybrid structure according to claim80, wherein at least one of the SP1 polypeptide monomers having an aminoacid sequence denoted by any one of SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:15 and SEQ IDNO:17.
 89. A self assembled hybrid structure comprising (a) a solidsubstrate having at least one nanopore perforating therethrough, and (b)at least one ring-like polypeptide situated at a region of said at leastone nanopore, said region being selected from an opening of the nanoporeand an interior region of said nanopore.
 90. A device comprising: (i) ahybrid structure comprising (a) a solid substrate having at least onenanopore perforating therethrough, and (b) at least one ring-likepolypeptide situated at a region of said at least one nanopore, saidregion being selected from an opening of the nanopore and an interiorregion of said nanopore, wherein the binding of the at least onering-like polypeptide to the nanopore being different from covalentbinding; and (ii) a measuring unit.
 91. The device according to claim90, wherein the at least one nanopore having an opening at a firstsurface of the substrate and a further opening at an opposite surface ofthe substrate, and interior surface spanning the opening at a firstsurface and the further opening at the opposite surface.
 92. The deviceaccording to claim 90, comprising an electrode assembly.
 93. The deviceaccording to claim 90, comprising a voltage source.
 94. A method foranalysis of at least one analyte in a sample comprising: (i) applying asample comprising at least one analyte or suspected to comprise at leastone analyte onto a hybrid structure, wherein the hybrid structurecomprising (a) a solid substrate having at least one nanoporeperforating therethrough, and (b) at least one ring-like polypeptidesituated at a region of said at least one nanopore, said region beingselected from an opening of the nanopore and an interior region of saidnanopore, wherein the binding of the at least one ring-like polypeptideto the nanopore being different from covalent binding, (ii) permittingthe sample to flow through the nanopore; (iii) determining at least oneof (a) presence or absence of an analyte in the sample, (b) identity ofthe analyte in the sample and (c) concentration of the analyte in thesample.
 95. The method according to claim 94, comprising monitoring atleast one measurable parameter related to the nanopore.
 96. A method forsequencing a nucleic acid molecule comprising: (i) applying the samplecomprising at least one nucleic acid molecule onto a hybrid structure,the hybrid structure comprising (a) a solid substrate having at leastone nanopore perforating therethrough, and (b) at least one ring-likepolypeptide situated at a region of said at least one nanopore, saidregion being selected from an opening of the nanopore and an interiorregion of said nanopore, wherein the binding of the at least onering-like polypeptide to the nanopore being different from covalentbinding, (ii) permitting the sample to flow through the nanopore; (ii)determining the sequence of the nucleic acid molecule.
 97. The methodaccording to claim 96, comprising permitting passing of at least onenucleotide of the nucleic acid molecule sequentially via the nanoporefrom one opening of the nanopore to an opposite opening of the nanoporeat each monitoring step.
 98. The method according to claim 96, whereinthe nucleic acid is single-stranded or double-stranded DNA or RNA.